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- EMDB-2277: Ribosome structures to near-atomic resolution from thirty thousan... -

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Basic information

Entry
Database: EMDB / ID: EMD-2277
TitleRibosome structures to near-atomic resolution from thirty thousand cryo-EM particles
Map dataReconstruction of 70S ribosome
Sample
  • Sample: T. thermophilus 70S ribosome
  • Complex: 70S ribosome
Keywordscryo-EM / single-particle analysis / direct electron detectors / ribosome / RELION / statistical movie processing
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsBai XC / Fernandez IS / McMullan G / Scheres SHW
CitationJournal: Elife / Year: 2013
Title: Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles.
Authors: Xiao-Chen Bai / Israel S Fernandez / Greg McMullan / Sjors H W Scheres /
Abstract: Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet ...Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.DOI:http://dx.doi.org/10.7554/eLife.00461.001.
History
DepositionJan 8, 2013-
Header (metadata) releaseJan 23, 2013-
Map releaseJan 23, 2013-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.24
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.24
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2277.jpg

Downloads & links

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Map

FileDownload / File: emd_2277.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 70S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 200 pix.
= 354. Å		1.77 Å/pix.
x 200 pix.
= 354. Å		1.77 Å/pix.
x 200 pix.
= 354. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24 / Movie #1: 0.24
Minimum - Maximum-0.58494776 - 1.0357604
Average (Standard dev.)0.00443429 (±0.06960686)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 354.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z354.000354.000354.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.5851.0360.004

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Supplemental data

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Sample components

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Entire : T. thermophilus 70S ribosome

EntireName: T. thermophilus 70S ribosome
Components
  • Sample: T. thermophilus 70S ribosome
  • Complex: 70S ribosome

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Supramolecule #1000: T. thermophilus 70S ribosome

SupramoleculeName: T. thermophilus 70S ribosome / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 3 MDa / Theoretical: 3 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 3 MDa / Theoretical: 3 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.45
Details: 3mM Hepes-KOH, 6.6 mM Tris-acetate pH 7.2, 3 mM NH4Cl, 6.6 mM NH4-acetate, 48 mM K-acetate, 4 mM Mg-acetate, 2.4 mM DTT
GridDetails: Quantifoil grids (2/2) with 3 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK II / Method: Blot 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateJul 1, 2012
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 360 / Average electron dose: 16 e/Å2
Details: Every image is the average of sixteen frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 79096 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.597 µm / Nominal defocus min: 1.178 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsUse a newly developed statistical movie processing to compensate for beam-induced movement
CTF correctionDetails: each image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: Use a newly developed statistical movie processing approach to compensate for beam-induced movement
Number images used: 35404

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Atomic model buiding 1

Initial modelPDB ID:

2wh1
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2wh2
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid body. The domains were separately fitted by manual docking using program Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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